1. Field of the Invention
Methods and products for regulating equilibrium or homeostasis between intestinal microflora and the immune system, especially via lymphoid tissue genesis associated with NOD1. Genesis or neogenesis of isolated lymphoid follicles (ILFs) using molecules that interact with NOD1 expressed on epithelial cells.
2. Description of the Related Art
Intestinal homeostasis between the intestinal microflora and the immune system is critical for efficient energy extraction from food and protection from pathogens by maintenance of a robust epithelial barrier. Homeostatic disruption can lead to an array of severe illnesses of major impact on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear.
After birth colonization of the intestines by commensal bacteria and other microorganisms evokes a response by the epithelial barrier and the immune system that induces recruitment of lymphocytes, maturation of lymphoid tissues, and production of IgA immunoglobulins. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells and include Peyer's patches, mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFS)1,2. Secondary lymphoid tissues, such as lymph nodes (LNs) and Peyer's patches (PPs), are induced to develop in the sterile environment of the fetus by lymphoid tissue inducer (LTi) cells3. After birth, hundreds of small clusters of LTi-like cells termed cryptopatches (CPs) form between crypts in the intestinal lamina propria4. During bacterial colonization, several lines of evidence indicate that CPs recruit B cells and develop into ILFs5,6, recapitulating the fetal development of LNs and PPs7. However, in contrast to the programmed development of LNs and PPs, the formation of mature ILFs from CPs requires induction by the gut flora1,8. Furthermore, in mice that harbor an expanded bacterial flora as a consequence of a lack of IgA, ILFs are abnormally large and numerous9. Given the inducible nature of ILFs, it is possible that these lymphoid tissues play an essential role in the equilibrium between the gut flora and the immune system7. However, the components of the gut flora that induce the genesis of ILFs, the factors that induce the recruitment of B cells into CPs, and the impact of ILFs on the gut flora remain largely unknown.
Methods for modulating NOD1 activity are disclosed by Girardin, et al., U.S. Pat. No. 7,078,165 which describes NOD1 as a pathogen recognition molecule expressed by eukaryotic cells that senses Gram-negative bacteria via a peptidoglycan motif, muramyl tripeptide or MTP. Girardin proposes to use MTP-like molecules to modulate NOD1 activity in eukaryotic cells.
Nuñez, et al., U.S. Pat. No. 7,244,557, relates to NOD1 protein as an intracellular signaling molecule and describes screening methods for molecules that modulate NOD1 activity using cells expressing NOD1.
Sansonetti, et al., U.S. Pat. No. 7,396,812, describes a method for enhancing host immune responses by administering MTP-like molecules, such as GlcNAc-MurNAc-L-Ala-D-Glu-mesoDAP (MTP), MurNAc-L-Ala-D-Glu-mesoDAP, and L-Ala-D-Glu-mesoDAP.
Nucleotide-binding oligomerization domain containing 1 (“NOD1”) is a gene found in humans and other mammals that encodes NOD1 protein. NOD1 protein contains a caspase activation and recruitment domain (CARD). Caspases or “cysteine-aspartic acid proteases” are a family of cysteine proteases that are involved in programmed cell death, apoptosis, cellular necrosis, and inflammation. Caspase recruitment domains are segments of proteins that interact with caspases and are found in a variety of different proteins, such as those involved in inflammation or apoptosis.
CCL20 is a small cytokine belonging to the CC chemokine family. It is known under various names including chemokine (C—C motif) ligand 20, liver activation regulated chemokine (LARC), or Macrophage Inflammatory Protein-3 (MIP3A). CCL20 is strongly chemotactic for different types of leukocytes and is implicated in the formation and function of mucosal lymphoid tissue, via chemoattraction of lymphocytes and dendritic cells towards the epithelial cells surrounding these tissues. CCL20 elicits its effects on its target cells by binding and activating chemokine receptor CCR6.
Murine β-defensin 3 (mBD3) is a defensin, one of a group of small cationic cysteine-rich cationic proteins found in both vertebrates and invertebrates. Beta-defensins exhibit broad spectrum antimicrobial activity against bacteria, fungi and enveloped viruses. They have molecular masses ranging from 2-6 kDa and have six to eight conserved cysteine residues. Whereas α-defensins are produced by phagocytes and Paneth cells, β-defensins are produced primarily by epithelial cells. Mouse β-defensin 3, the ortholog of human β-defensin 2, binds to (is a ligand of) CCR6; Biragyn, et al., J Immunol. Dec. 1, 2001; 167(11):6644-53. The structure of human β-defensin 2 (also known as DEFB2, DEFB4) is incorporated by reference to UniProtKB/Swiss-Prot O15263 (DEFB2_HUMAN) version 94, Mar. 3, 2009. The structure of murine β-defensin 3 is incorporated by reference to UniProtKB/Swiss-Prot Q9WTL0 (DEFB3_MOUSE), version 60, Mar. 3, 2009.